Mutations in gene regions may cause mis-sense mutations accompanying changes in translated amino acids, silent mutations without any change in the amino acids, and frame shift mutations, wherein the translation frame is shifted due to deletion or insertion of nucleotide(s). Non-sense mutations, wherein a stop codon is generated at incorrect location, are also included as mis-sense mutations. In addition, mutations in gene regions also have the potential of leading to gene translation abnormalities through splicing abnormalities and such. Many of these mutations, other than silent mutations, are accompanied by structural or functional changes in translated proteins.
Moreover, abnormalities in expression regulatory regions have the risk of affecting the expression regulatory mechanism of proteins.
Among differences in the nucleotide sequence of nucleic acids, mutations that are present at a frequency of 1% or more in a certain population are particularly referred to as polymorphisms. A population refers to a population that can be distinguished by geographical isolation or subspecies. For example, even in the case of a mutation that occurs at a frequency of less than 1% in Japanese, if that mutation is found at a frequency of 1% or more among other races, it is not a mutation, but rather a polymorphism.
Among these polymorphisms, polymorphisms due to insertion, deletion, or displacement of a single nucleotide are particularly referred to as single nucleotide polymorphisms (hereinafter, abbreviated as SNPs). SNPs are of high profile because these are mutations most frequently appearing in the human genome.
Since polymorphisms are spread throughout a population at a fixed frequency, they are considered not to accompany any changes in phenotypes or only changes of such phenotypes that influence phenotypes called constitution, and not those that are particularly disadvantageous for survival (reproduction). For example, predispositions to typical adult diseases, such as diabetes, rheumatism, allergies, autoimmune diseases, obesity, and cancer, are suggested to be determined by polymorphisms when the disease is governed by genetic characteristics. Further, drug metabolism, human leukocyte histocompatibility antigens (hereinafter abbreviated as HLA), and such are also governed by polymorphisms. Moreover, the majority of these polymorphisms are revealed to be SNPs.
Since polymorphisms have these characteristics, like the microsatellite polymorphisms, they are used to determine disease-associated genes by chromosome mapping, linkage analysis, and such. SNPs are present at a rate of one every 300 to 600 nucleotides. Thus, use of SNPs enables construction of a detailed map which is expected to facilitate gene determination.
Moreover, much information is expected to be obtainable by gathering information relating to polymorphism locations and fluctuations, and analyzing their correlation with certain phenotypes. For example, side effects of a drug may be prevented through the discovery of SNPs related to such side effects of the drug. By overcoming these side effects, numerous drugs whose practical application has been abandoned may be reevaluated as safe drugs.
In the case of bacteria and viruses, the subtypes of hepatitis C virus (hereinafter abbreviated as HCV) and such are classifications based on the characteristics of a nucleotide sequence commonly found at a fixed ratio, and thus, these subtypes and such also can be considered as polymorphisms. Mutation analyses investigating the genotypes of such polymorphisms are referred to as typing.
Therapeutic efficacy of α-interferon on HCV differs according to particular subtypes. Therefore, typing provides important information useful in selecting methods for the treatment. Further, pathogens beside HCV, such as influenza virus, malaria pathogens, and Helicobacter pylori, are also demonstrated to be pathogens with different therapeutic efficacy according to the subtypes. Thus, typing provides important information for determining the mode of treatment for these pathogens, too.
In contrast to polymorphisms, mutations found at a ratio less than 1% are mutations that do not spread throughout a population in the case of humans, and nearly all of them can be mentioned as mutations involved in some kind of disease. Specifically, these mutations correspond to those found in hereditary diseases. In addition, some of the mutations found in individuals are also associated with disease, such as mutations found in association with cancer and so on. The detection of such mutations provides decisive information in diagnosing the corresponding disease.
Whether or not the nucleotide sequence of a certain gene differs from the predicted nucleotide sequence can be confirmed by hybridization of the complementary nucleotide sequence. More specifically, hybridization with a primer or probe is used.
For example, PCR primers are only able to act as primers when the target nucleotide sequence has a nucleotide sequence that is complementary to the primer. Based on this principle, a target nucleotide sequence can be examined to determine whether or not it is complementary to the primer, using the PCR amplification product as an indicator. However, there are several problems with this method of confirming nucleotide sequences based on PCR. First, the checking mechanism of the nucleotide sequence by the primer is incomplete. Methods for detecting single nucleotide differences by amplification reaction of PCR methods have been considered (allele-specific PCR, Nucleic Acids Res. 17: p. 2503, 1989; Genomics 5: p. 535, 1989; J. Lab. Clin. Med. 114: p. 105, 1989). However, as was reported by S. Kwok et al. (Nucleic Acids Res. 18: p. 999, 1990) when using a primer with a single nucleotide difference, the reaction proceeds at about 0.1% to 85% (which differs depending on the difference in the sequence) per amplification cycle as compared with a completely complementary primer. In other words, complementary strand synthesis frequently occurs even if the nucleotide sequence is not completely complementary with the primer. Consequently, in order to distinguish single nucleotide differences using allele-specific PCR, artificial insertion of another mismatch at another location has been shown to be necessary. However, in this case as well, conditions must be precisely set according to the sequence difference, which has not become a general technique. Thus, it is difficult to identify single nucleotide differences, such as SNPs, by PCR under ordinary conditions.
In other words, methods wherein nucleotide sequences with slight differences are detected based on hybridization of a complementary nucleotide sequence have the potential of being inaccurately detected. According to the PCR method, products formed by inaccurate detection do also function as complete templates, and thus, these products trigger an exponential amplification. The problem of using conventional PCR method to detect slight nucleotide sequence is that, despite the possibility of inaccurate detection, a high degree of amplification occurs based on the inaccurate detection.
The second problem of the PCR method is that primers for only two regions can be configured due to the principle. Thus, when a plurality of genes composed of similar nucleotide sequences are present in the same sample, mutations and polymorphisms of the genes is extremely difficult when the presence or absence of amplification products based on PCR is used an indicator. This is because the PCR reaction proceeds when the primer hybridizes to one of the genes, even if a gene with mutation exists in the sample.
For example, a primer for detecting a mutation of a certain gene in a family gene, such as human CYP2C19, acts as a primer on other genes as well. FIG. 6 depicts mutually similar nucleotide sequences found in the human CYP2C19 family. As suggested from the Figure, a primer for detecting mutation of a certain gene also acts as a primer for other genes of the wild type. Under such conditions, it is difficult to simultaneously identify the similar gene and detect the mutation with only two regions.
To solve the two problems described above, so called PCR-dependent mutation detection techniques have been reported wherein the target gene is specifically amplified by PCR, and then the mutations are detected using a probe or primer. Among these techniques, the DNA chip method is a detection technique which is particularly attracting attention. A large number of similar nucleotide sequences can be arranged in minute compartments on a DNA chip. By controlling the reaction environment of the fine spaces on the DNA chip, the presence or absence of hybridization based on slight differences of the nucleotide sequences can be detected. However, the reproducibility of analyzed data obtained using DNA chip is a major problem. The hybridization conditions on the DNA chip must be precisely set, due to the fine reaction spaces. In order to maintain a fixed level of reproducibility, sophisticated techniques and utmost caution are required. Moreover, since it is a PCR dependent technique, two steps are required; the high price of a DNA chip is a further problem that needs to be resolved.
On the other hand, the Invader method (Mol. Diagn. 4: p. 135, 1999) and the RCA method (Nat. Genet. 19: p. 225, 1998) have been reported as examples of PCR-independent mutation detection techniques. However, the reaction specificity is dependent on the probe set of two adjacent regions for both of these methods. As was pointed out with respect to PCR, it is difficult to simultaneously identify similar genes and detect mutations with only two regions.
As has been described above, known methods for confirming nucleotide sequences have problems. For example, accurate identification of slight nucleotide sequences cannot be performed in a single step. Also, the methods encounter difficulty in simultaneously identifying similar genes and detecting mutations. Further, even if it is technically possible to identify similar genes and detect mutations, as with PCR-dependent technology, it is either difficult to maintain accuracy or a complex procedure is required, due to the need for a plurality of steps. Finally, improvement in economic aspects is desired as well.